Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. Moreover, it is not clear how different subcellular localizations influence a signaling protein's function. The current grant is focused on understanding the mechanism that govern assembly, membrane localization, and intracellular trafficking of heterotrimeric G proteins, and, in addition, is determining the role of G proteins at non-canonical locations in the cell, such as a role for G[unreadable]? in regulating vesicle transport at Golgi membranes. The Revision application will expand the scope of these studies by investigating mechanisms of membrane localization for G protein-coupled receptor kinase 5 (GRK5). G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs. Phosphorylation of activated GPCRs by GRKs plays a critical role in GPCR desensitization;phosphorylation promotes recruitment of arrestins to the GPCR which prevents further activation of heterotrimeric G proteins and promotes the internalization of the GPCR. Thus, GRKs are universally important for regulating signaling mediated by GPCRs and G protein pathways. Because of the ubiquitous importance of GPCR signaling in regulating a vast number of physiological functions, GRKs have essential roles in the proper regulation of a multitude of physiological processes that are initiated by extracellular signals. Work in this laboratory has recently provided data to support a novel mechanism that regulates membrane targeting of GRK5: dimerization of GRK5 is critical for its plasma membrane localization. This application will focus on defining this new model for membrane localization of GRK5. To address this goal, the major objectives of the proposal are 1) test the hypothesis that GRK5 forms dimers in cells;and 2) test the hypothesis that dimerization of GRK5 is critical for plasma membrane localization and function. These objectives will be pursued by a variety of experimental approaches, including expression of recombinant proteins in cultured cells, fluorescence and immunofluorescence microscopy localization of proteins, immunoblotting, immunoprecipitations, bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer (BRET), inducible homodimerization systems, G protein-mediated signaling assays in intact cells, and phosphorylation assays. PUBLIC HEATH RELEVANCE: G protein-mediated signaling pathways regulate numerous physiological responses, including cardiovascular function, neurotransmitter responses, cell differentiation, cell migration, immune cell function, and smell, taste, and vision;and dysregulation of G protein-mediated signaling pathways contribute to numerous disease states, including heart disease, hypertension, cancer, metastasis, endocrine disorders, brain disorders and blindness. G protein-coupled receptor (GPCR) kinases (GRKs) are key proteins involved in turning-off a signaling response to insure that the response is of the proper duration. To function properly, the GRKs must interact tightly with cellular membranes. Blocking membrane recruitment of one GRK, called GRK2, has been used as an effective treatment for heart disease in animal models. GRK5, the focus of the research project, is important in neuronal, as well as cardiovascular and other responses. Thus, it is clear that understanding the mechanisms that regulate GRK localization could lead to novel ways to therapeutically target individual GRKs.